Active-site engineering of ω-transaminase from Ochrobactrum anthropi for preparation of L-2-aminobutyric acid.

BACKGROUND: The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering. RESULTS: Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g. CONCLUSION: This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.

A novel global transcriptional perturbation target identified by forward genetics reprograms Vibrio natriegens for improving recombinant protein production.

Vibrio natriegens is known to be the fastest-growing free-living bacterium with the potential to be a novel protein expression system other than Escherichia coli. Seven sampled genes of interest (GOIs) encoding biocatalyst enzymes, including Ochrobactrum anthropi-derived ω-transaminase (OATA), were strongly expressed in E. coli but weakly in V. natriegens using the pET expression system. In this study, we fused the C-terminal of OATA with green fluorescent protein (GFP) and obtained V. natriegens mutants that could increase both protein yield and enzyme activity of OATA as well as the other three GOIs by ultraviolet mutagenesis, fluorescence-activated cell sorting (FACS), and OATA colorimetric assay. Furthermore, next-generation sequencing and strain reconstruction revealed that the Y457 variants in the conserved site of endogenous RNA polymerase (RNAP) ß' subunit rpoC are responsible for the increase in recombinant protein yield. We speculated that the mutation of rpoC Y457 may reprogram V. natriegens's innate gene transcription, thereby increasing the copy number of pET plasmids and soluble protein yield of certain GOIs. The increase in GOI expression may partly be attributed to the increase in copy number. In conclusion, GOI-GFP fusion combined with FACS is a powerful tool of forward genetics that can be used to obtain a superior expression chassis. If more high-expression-related targets are found for more GOIs, it would make the construction of next-generation protein expression chassis more time-saving.

Genomic and biotechnological insights on stress-linked polyphosphate production induced by chromium(III) in Ochrobactrum anthropi DE2010.

The resistance of microorganisms to heavy metals in polluted environments is mediated by genetically determined mechanisms. One such mechanism includes the intracellular sequestration of heavy metals in polyphosphate (polyP) inclusions. In Cr(III) contaminated mediums, Ochrobactrum anthropi DE2010 is able to bind and sequester Cr(III) in polyP inclusions. In order to further study the relationship between Cr(III) tolerance and polyP production in O. anthropi DE2010, we carried out whole genomic sequencing, analysis of single nucleotide polymorphisms (SNPs), polyP chemical quantification, and determination of the relative abundance and morphometry of polyP inclusions. In the O. anthropi DE2010 genome, six polyP and pyrophosphate (PPi) metabolic genes were found. Furthermore, genomic analysis via SNPs calling revealed that O. anthropi ATCC49188 and DE2010 strains had average variations of 1.51% in their whole genome sequences and 1.35% variation associated with the principal polyP metabolic gene cluster. In addition, the accumulation of polyP in the DE2010 strain and number of polyP inclusions found were directly correlated with the concentration of Cr(III) in contaminated cultures. The results presented in this study may enhance the understanding of polyP production in response to Cr(III) toxicity in the O. anthropi DE2010 strain. This knowledge may facilitate the successful removal of Cr(III) from the natural environment.

Improvement of whole-cell transamination with Saccharomyces cerevisiae using metabolic engineering and cell pre-adaptation.

BACKGROUND: Whole-cell biocatalysis based on metabolically active baker's yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels. RESULTS: Herein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5'-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using L-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions. CONCLUSIONS: Altogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion.

Microbial associates of the southern mole cricket (Scapteriscus borellii) are highly pathogenic.

We report the isolation and identification of seven bacterial strains and one fungal strain from dead and diseased Scapteriscus borellii mole crickets collected from a golf course in southern California. Using 16S and 18S rRNA gene sequence analysis we identified the microbes as Serratia marcescens (red), S. marcescens (white), S. marcescens (purple), Achromobacter xylosoxidans, Chryseobacterium sp., Ochrobactrum anthropi, Tsukamurella tryosinosolvens, and Beauveria bassiana. We performed a dose response curve for each of these cricket-associated microbial strains (except T. tryosinosolvens) and two other strains of S. marcescens (DB1140 and ATCC 13880). We found that all of these microbes except O. anthropi were highly pathogenic to D. melanogaster compared to the other strains of S. marcescens. Injecting the mole cricket associated strains of Serratia into flies killed all infected flies in ≤24h. For all other strains, the median time to death of injected flies varied in a dose-dependent manner. In vivo growth assessments of these microbes suggested that the host immune system was quickly overcome. We used disease tolerance curves to better understand the host-microbe interactions. Further studies are necessary to understand in mechanistic detail the virulence mechanisms of these mole cricket associated microbes and how this association may have influenced the evolution of mole cricket immunity.

Utility of Ochrobactrum anthropi YC152 in a Microbial Fuel Cell as an Early Warning Device for Hexavalent Chromium Determination.

Fast hexavalent chromium (Cr(VI)) determination is important for environmental risk and health-related considerations. We used a microbial fuel cell-based biosensor inoculated with a facultatively anaerobic, Cr(VI)-reducing, and exoelectrogenic Ochrobactrum anthropi YC152 to determine the Cr(VI) concentration in water. The results indicated that O. anthropi YC152 exhibited high adaptability to pH, temperature, salinity, and water quality under anaerobic conditions. The stable performance of the microbial fuel cell (MFC)-based biosensor indicated its potential as a reliable biosensor system. The MFC voltage decreased as the Cr(VI) concentration in the MFC increased. Two satisfactory linear relationships were observed between the Cr(VI) concentration and voltage output for various Cr(VI) concentration ranges (0.0125-0.3 mg/L and 0.3-5 mg/L). The MFC biosensor is a simple device that can accurately measure Cr(VI) concentrations in drinking water, groundwater, and electroplating wastewater in 45 min with low deviations (<10%). The use of the biosensor can help in preventing the violation of effluent regulations and the maximum allowable concentration of Cr(VI) in water. Thus, the developed MFC biosensor has potential as an early warning detection device for Cr(VI) determination even if O. anthropi YC152 is a possible opportunistic pathogen.

Development of a loop-mediated isothermal amplification method for the rapid detection of the dioxin-degrading bacterium Ochrobactrum anthropi in soil.

In this study, loop-mediated isothermal amplification (LAMP) and real-time LAMP assays were developed to detect the dioxin-degrading bacterium Ochrobactrum anthropi strain BD-1 in soil. Four primers were designed to use ITS gene amplification for the strain O. anthropi BD-1. The real-time LAMP assay was found to accomplish the reaction by 1 pg of genomic DNA load when used for nucleic acid amplification. This assay was then applied to detect O. anthropi BD-1 in eight soil samples collected from a dioxin-contaminated site. The results demonstrated that these newly developed LAMP and real-time LAMP assays will not only be useful and efficient tools for detecting the target gene, but also be used as molecular tools for monitoring the growth of dioxin-degrading O. anthropi in the soil. This is the first report to demonstrate the use of LAMP assays to monitor the presence of O. anthropi in dioxin-contaminated soil. The application of this method should improve the biomonitoring of dioxin contamination.

[Research progress of Ochrobactrum anthropic--A review].

Ochrobactrum anthropi is a gram-negative bacillus, usually known as an opportunistic pathogen. Mostly its infection is related with systemic or local lower immunity, and manifested as bacteremia, meningitis, purulent infection. Recently, along with expanded infection, it has become an important human pathogen. The prevention, clinical diagnose and treatment become complicated because varied clinical symptoms increased antibiotic resistance and cross immune reaction with others pathogens. In this review, we summarized the biological characteristics, differential diagnosis, immunity, resistance and genomic characteristics of Ochrobactrum anthropi, to provide reference for prevention, control and treatment management of this disease.

Typing of Ochrobactrum anthropi clinical isolates using automated repetitive extragenic palindromic-polymerase chain reaction DNA fingerprinting and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

BACKGROUND: Ochrobactrum anthropi (O. anthropi), is a non-fermenting gram-negative bacillus usually found in the environment. Nevertheless, during the past decade it has been identified as pathogenic to immunocompromised patients. In this study, we assessed the usefulness of the automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR-based DiversiLab™ system, bioMèrieux, France) and of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF MS) for typing of twentythree O. anthropi clinical isolates that we found over a four-months period (from April 2011 to August 2011) in bacteriemic patients admitted in the same operative unit of our hospital. Pulsed-field gel electrophoresis (PFGE), commonly accepted as the gold standard technique for typing, was also used. Analysis was carried out using the Pearson correlation coefficient to determine the distance matrice and the unweighted pair group method with arithmetic mean (UPGMA) to generate dendogram. RESULTS: Rep-PCR analysis identified four different patterns: three that clustered together with 97% or more pattern similarity, and one whose members showed < 95% pattern similarity. Interestingly, strains isolated later (from 11/06/2011 to 24/08/2011) displayed a pattern with 99% similarity. MALDI-TOF MS evaluation clustered the twentythree strains of O. anthropi into a single group containing four distinct subgroups, each comprising the majority of strains clustering below 5 distance levels, indicating a high similarity between the isolates. CONCLUSIONS: Our results indicate that these isolates are clonally-related and the methods used afforded a valuable contribution to the epidemiology, prevention and control of the infections caused by this pathogen.

Clinical characteristics of Ochrobactrum anthropi bacteremia.

The clinical picture of Ochrobactrum anthropi infection is not well described because the infection is rare in humans and identification of the pathogen is difficult. We present a case of O. anthropi bacteremia that was initially misidentified as Ralstonia paucula and later identified by 16S rRNA sequencing and recA analysis.

Biocontrol potential of phylloplane bacterium Ochrobactrum anthropi BMO-111 against blister blight disease of tea.

AIMS: The present study was carried out to screen the phylloplane bacteria from tea for antagonism against grey blight caused by Pestalotiopsis theae and blister bight caused by Exobasidium vexans and to further evaluate the efficient isolates for disease control potential under field condition. METHODS AND RESULTS: A total of 316 morphologically different phylloplane bacteria were isolated. Among the antagonists, the isolates designated as BMO-075, BMO-111 and BMO-147 exhibited maximum inhibitory activity against both the pathogens under in vitro conditions and hence were selected for further evaluation under microplot field trial. Foliar application of 36-h-old culture of BMO-111 (1 × 10(8) colony-forming units ml(-1) ) significantly reduced the blister blight disease incidence than the other isolates. The culture of BMO-111 as well as its culture filtrate effectively inhibited the mycelial growth of various fungal plant pathogens. The isolate BMO-111 was identified as Ochrobactrum anthropi based on the morphological and 16S rDNA sequence analyses. CONCLUSIONS: It could be concluded that the biocontrol agent O. anthropi BMO-111 was effective against blister blight disease of tea. SIGNIFICANCE AND IMPACT OF THE STUDY: Further study is required to demonstrate the mechanism of its action and formulation for the biocontrol potential against blister blight disease of tea.

Group X aldehyde dehydrogenases of Pseudomonas aeruginosa PAO1 degrade hydrazones.

Hydrazones are natural and synthetic compounds containing a C=N-N moiety. Here we found that the opportunistic pathogen Pseudomonas aeruginosa PAO1 produced NAD(+)- or NADP(+)-dependent hydrazone dehydrogenase (HDH), which converts hydrazones to the corresponding hydrazides and acids rather than to the simple hydrolytic product aldehydes. Gene cloning indicated that the HDH is part of the group X aldehyde dehydrogenase (ALDH) family, which is distributed among bacteria, although the physiological roles of the ALDH family remain unknown. The PAO1 strain upregulated HDH in the presence of the hydrazone adipic acid bis(ethylidene hydrazide) (AEH). Gene disruption of the HDH-encoding hdhA (PA4022) decreased growth rates in culture medium containing AEH as the sole carbon source, and this effect was more obvious in the double gene disruption of hdhA and its orthologous exaC (PA1984), indicating that these genes are responsible for hydrazone utilization. Recombinant proteins of group X ALDHs from Escherichia coli, Paracoccus denitrificans, and Ochrobactrum anthropi also acted as HDHs in that they produced HDH activity in the cells and degraded hydrazones. These findings indicated the physiological roles of group X ALDHs in bacteria and showed that they comprise a distinct ALDH subfamily.

Enzymatic properties of the glycine D-alanine [corrected] aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji).

The gdaA gene encoding S12 family glycine-D-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the D-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high D-stereospecificity and efficiently released N-terminal glycine and D-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and D-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and D-alanine aminopeptidase activity detected at a pH range of 6 to 9.

Distribution of glyphosate and methylphosphonate catabolism systems in soil bacteria Ochrobactrum anthropi and Achromobacter sp.

Bacterial strains capable of utilizing methylphosphonic acid (MP) or glyphosate (GP) as the sole sources of phosphorus were isolated from soils contaminated with these organophosphonates. The strains isolated from MP-contaminated soils grew on MP and failed to grow on GP. One group of the isolates from GP-contaminated soils grew only on MP, while the other one grew on MP and GP. Strains Achromobacter sp. MPS 12 (VKM B-2694), MP degraders group, and Ochrobactrum anthropi GPK 3 (VKM B-2554D), GP degraders group, demonstrated the best degradative capabilities towards MP and GP, respectively, and were studied for the distribution of their organophosphonate catabolism systems. In Achromobacter sp. MPS 12, degradation of MP was catalyzed by C-P lyase incapable of degrading GP (C-P lyase I). Adaptation to growth on GP yielded the strain Achromobacter sp. MPS 12A, which retained its ability to degrade MP via C-P lyase I and was capable of degrading GP with formation of sarcosine, thus suggesting the involvement of a GP-specific C-P lyase II. O. anthropi GPK 3 also degraded MP via C-P lyase I, but degradation of GP in it was initiated by glyphosate oxidoreductase, which was followed by product transformation via the phosphonatase pathway.

Molecular determinants for substrate selectivity of ω-transaminases.

ω-Transaminase (ω-TA) is an industrially important enzyme for production of chiral amines. About 20 (S)-specific ω-TAs known to date show remarkably similar substrate selectivity characterized by stringent steric constraint precluding entry of a substituent larger than an ethyl group in the small binding pocket (S) and dual recognition of an aromatic substituent as well as a carboxylate group in the large pocket (L). The strictly defined substrate selectivity of the available ω-TAs remains a limiting factor in the production of structurally diverse chiral amines. In this work, we cloned, purified, and characterized three new ω-TAs from Ochrobactrum anthropi, Acinetobacter baumannii, and Acetobacter pasteurianus that were identified by a BLASTP search using the previously studied ω-TA from Paracoccus denitrificans. All the new ω-TAs exhibited similar substrate specificity, which led us to explore whether the molecular determinants for the substrate specificity are conserved among the ω-TAs. To this end, key active site residues were identified by docking simulation using the X-ray structure of the ω-TA from Pseudomonas putida. We found that the dual recognition in the L pocket is ascribed to Tyr23, Phe88*, and Tyr152 for hydrophobic interaction and Arg414 for recognition of a carboxylate group. In addition, the docking simulation indicates that Trp60 and Ile262 form the S pocket where the substituent size up to an ethyl group turns out to be sterically allowed. The six key residues were found to be essentially conserved among nine ω-TA sequences, underlying the molecular basis for the high similarity in the substrate selectivity.

Whole-genome analysis and description of an IMP-8-producing Ochrobactrum anthropi.

OBEJECTIVES: To explore the genomic characterization of an IMP-8-producing Ochrobactrum anthropic and give suggestions for the application of antibiotics. METHODS: In 2021, the infection caused by CRKP was under control after nearly three months of using CAV, however, carbapenem-resistant O. anthropi isolates were collected from a rectal swab sample of a patient with Lumbar Disc Herniation Postoperative Infection. The rectal swab was then enriched in lysogeny broth overnight and inoculated onto China Blue agar plates containing 0.3µg/mL meropenem. And we investigated the characteristics of this carbapenem-resistant O. anthropi by MALDI-TOF MS, Immune colloidal gold technique, conjugation experiment, whole genome sequencing and antimicrobial susceptibility testing. RESULTS: Antimicrobial susceptibility testing showed that the O. anthropi were resistant to imipenem, cefmetazole, ceftazidime, cefotaxime, piperacillin/tazobactam, sulbactam/cefopcrazone, ceftazidime/avibactam, cefepime, ciprofloxacin, aztreonam, and not susceptible to meropenem, ertapenem, polymyxin B, tigecycline, amikacin. Immune colloidal gold technique reflected that this strain produced IMP carbapenemases, and the presence of IMP-8 was verified by WGS, which was located in a 21,442 bp, nonconjugative plasmid. CONCLUSION: Improper antibiotic treatment can cause intestinal flora imbalance and even bacteremia in patients, we should use antibiotics wisely and develop individualized treatment options.

Genome of Ochrobactrum anthropi ATCC 49188 T, a versatile opportunistic pathogen and symbiont of several eukaryotic hosts.

Ochrobactrum anthropi is a common soil alphaproteobacterium that colonizes a wide spectrum of organisms and is being increasingly recognized as an opportunistic human pathogen. Potentially life-threatening infections, such as endocarditis, are included in the list of reported O. anthropi infections. These reports, together with the scant number of studies and the organism's phylogenetic proximity to the highly pathogenic brucellae, make O. anthropi an attractive model of bacterial pathogenicity. Here we report the genome sequence of the type strain O. anthropi ATCC 49188, which revealed the presence of two chromosomes and four plasmids.

Environmental microbiota represents a natural reservoir for dissemination of clinically relevant metallo-beta-lactamases.

A total of 10 metallo-ß-lactamase-producing isolates of six different species, including Brevundimonas diminuta (n = 3), Rhizobium radiobacter (n = 2), Pseudomonas monteilii (n = 1), Pseudomonas aeruginosa (n = 2), Ochrobactrum anthropi (n = 1), and Enterobacter ludwigii (n = 1), were detected in the sewage water of a hospital. The presence of bla(VIM-13) associated with a Tn1721-class 1 integron structure was detected in all but one of the isolates (E. ludwigii, which produced VIM-2), and in two of them (R. radiobacter), this structure was located on a plasmid, suggesting that environmental bacteria represent a reservoir for the dissemination of clinically relevant metallo-ß-lactamase genes.